Regulatory role of VvsB protein on serine protease activity of VvsA in Vibrio vulnificus

Abstract Background: Vibrio vulnificus NCIMB2137, a Gram-negative, metalloprotease negative estuarine strain was isolated from a diseased eel. A 45 kDa chymotrypsin-like alkaline serine protease known as VvsA has been recently reported as one of the major virulence factor responsible for the pathogenesis of this strain. The vvsA gene along with a downstream gene vvsB, whose function is still unknown constitute an operon designated as vvsAB. Objective: This study examines the contribution of VvsB to the functionality of VvsA. Method: In this study, VvsB was individually expressed using Rapid Translation System (RTS system), followed by an analysis of its role in regulating the serine protease activity of VvsA. Result: The proteolytic activity of VvsA increased upon the addition of purified VvsB to the culture supernatant of V. vulnificus. However, the attempts of protein expression using an E. coli system revealed a noteworthy observation that protein expression from the vvsA gene exhibited higher protease activity compared to that from the vvsAB gene within the cytoplasmic fraction. These findings suggest an intricate interplay between VvsB and VvsA, where VvsB potentially interacts with VvsA inside the bacterium and suppress the proteolytic activity. While outside the bacterial milieu, VvsB appears to stimulate the activation of inactive VvsA. Conclusion: The findings suggest that Vibrio vulnificus regulates VvsA activity through the action of VvsB, both intracellularly and extracellularly, to ensure its survival.


Introduction
Pr oteases pr oduced by bacteria exhibit toxicity by degrading host proteins at the site of infection and by disrupting the host signaling system (Miyoshi andShinoda 2000 , Shinoda andMiyoshi 2011 ).Ho w e v er, these pr oteases also pose a thr eat to bacterial cells themselves, as they degrade cellular constituents .T hus , it becomes crucial for bacteria not only to regulate the production r ate of pr oteases but also to expr ess their activity at specific sites.
Ther efor e, it is imper ativ e to devise methods that prevent the produced proteases from decomposing cellular components.Howe v er, pr otease expr ession le v els ar e typicall y low in bacteria, and the intricate regulatory mechanisms governing the expression of active bacterial proteases remain incompletely understood.
Vibrio vulnificus is a pr e v alent estuarine bacterium c har acterized by its Gr am-negativ e and halophilic nature.Despite its common occurrence, this bacterium transforms into an opportunistic human pathogen, demonstrating high lethality and being implicated in seafood-related fatalities across various countries (Jones and Oliver 2009 ).The resultant primary septicemia from V. vulnificus infection is particularl y deadl y, with an av er a ge mortality r ate surpassing 50%.
The potential virulence factor termed as VvsA, a serine protease, has been reported in V. vulnificus strains isolated from a diseased eel (Tison et al. 1982, Senoh et al. 2005, Wang et al. 2008 ).VvsA is demonstrated to be a chymotrypsin-like protease.It has been shown that 45 kDa mature VvsA is deriv ed fr om the 59 kDa intermediate product after removal of the C-terminal 14 kDa polypeptide .T his VvsA is an orthologue of an extr acellular pr otease produced by Vibrio parahaemolyticus , a causative agent responsible for both human wound infection as well as gastroenteritis (Chakraborty et al. 1997 ).Hence, it is reasonable to infer that VvsA may play a role in the distinctive skin damage observed in human infections.Additionally, it could serve as a virulence factor in eel vibriosis, c har acterized by both external and internal hemorrhages (Lee et al. 2002 ).VvsA is encoded in an operon which contain two genes vvsA downstream gene vvsB .The vvsA and vvsB were separated by only 6 bp.The homologue of VvsB precursor (115aa) was found in several Vibrio species of the serine pr otease famil y.This identity w as lo w (36.5-56.5%).The 9.1 kDa VvsB was derived by removing of the C-terminal 2.9 kDa signal peptide.But its function is not yet known (Miyoshi et al. 2012 ).
In r ecent times, cell-fr ee pr otein synthesis systems hav e emerged as a po w erful technology platform for the r a pid, efficient, and cost-effectiv e anal yses of actions of pr oteins (Sc hwarz et al. 2008 ).Sometimes it is being difficult to express some bacterial proteins in living cells because of their potent toxicity.So, the in vitro protein synthesis machinery is a technical boon to overcome such limitations.
This study aims to investigate the role of VvsB in regulating the functionality and activity of VvsA, a 45 kDa chymotrypsin-like al-Table 1. Primers used in this study.

Primer
Sequence (5' → 3') GGGCGAA TTG GAGCTC TTCA T AGAACT A TCTTCTT A TGTTTTG Underlined: Ov erla pping r egion Bold: The restriction enyme site on the vector multiple-cloning site (MCS) kaline serine protease, in Vibrio vulnificus NCIMB2137.Specifically, the r esearc h seeks to understand how VvsB influences the pr oteolytic activity of VvsA both within the bacterial cell and in the extr acellular envir onment, ther eby contributing to the pathogen's virulence and survival.

Bacterial strains and growth conditions
Vibrio vulnificus NCIMB2137 was isolated from eel.The bacteria wer e gr own in Luria-Bertani medium (1.0%Bacto tryptone, 0.5% Bacto yeast extract, 1.0% NaCl) at 37 • C with appropriate antibiotics .T he genomic DNA of V. vulnificus was extracted as described earlier (Senoh et al. 2005 ).

T he DN A templa te for cloning of vvsB gene encoding serine proteases for RTS system
The r a pid tr anslation system (RTS100 E. coli HY Kit, 5PRIME Inc., Gaithersburg, MD, USA) was used as a cell-free translation system for the expression of proteins in vitro .In this study, a linear DNA fr a gment ( vvsB ) inserted into the RTS expr ession v ector pIVEX2.4d(RTS TM pIVEX His-tag 2 nd Generation Vector Set Short Instruction 5PRIME Inc., Gaithersburg, MD, USA) to generate a hybrid plasmid.Genomic DNA of Vibrio vulnificus NCIMB2137 strain was used as a PCR template for the amplification of the vvsB gene (GenBank accession number, AB509375), using primers listed in Table 1 .
Next, PCR amplified vvsB (282 bp) gene without a signal peptide sequence was cloned in the expression vector pIVEX2.4d(Kawase et al. 2022 ).It was then transformed into E. coli DH5 α strain and the positiv e tr ansformants wer e selected on ampicillin supplemented LB plates .T he recombinant plasmid was purified using Quantum Prep Plasmid mini Prep (Bio-Rad Laboratories , Hercules , C A, USA).

In vitro synthesis of proteins by RTS
The synthesis of serine protease was done using circular DNA (0.5 μg) expressed in in vitro system.The 50 μl reaction solution was set up according to manufacturer's protocol (RTS100 E. coli HY kit, 5PRIME, USA).The coupled tr anscription-tr anslation r eaction was carried in a RTS ProteoMaster (Roche) at 25ºC, 400 rpm for 6 h.The protease inhibitor was added only if metalloprotease was expressed.The serine protease inhibitor used was the cOmplete, Mini (Roche & Sigma Aldrich Japan).

Purification of His-tagged protein
For purification of the RTS pr oduct, Ca ptur em His-ta gged Purification Mini prep Kit (TaKaRa, J apan) w as used.200 μl of the r eaction mixtur e w as purified accor ding to the manufacture's protocol.

Assay for protease
Azocasein at a concentration of 5 mg/ml served as the substrate for assessing protease activity.The protease activities of RTS products were evaluated using the procedure outlined by Miyoshi et al. ( 1987 ).Absorbance readings were taken at 440 nm, with one protease unit (PU) defined as the quantity of enzyme capable of hydr ol yzing 1 μg of azocasein within 1minute.Protein amounts was measured by using a spectrophotometer.

N-terminal amino acid sequence
The proteins separated by SDS-PAGE were transferred to a PVDF membrane (BIO-RAD, USA) and stained with Amido Black T. Nterminal amino acid sequence was determined using an Applied Biosystem Precise Sequencer (Applied Biosystem, Foster City, CA, U.S.A.)

Purification of VvsB synthesized by RTS system
The protein synthesis was performed using circular vvsB DNA (Fig. 1 A) in RTS system at 25 • C for 6 h.The RTS reaction mixture was applied to His-tag column to get purified VvsB.In Fig. 2 , lane 3 sho w ed VvsB purification done by His-tag column after syn- F igure 2. SDS-PAGE follo w ed b y Coomassie brilliant blue staining of the gel.The protein produced by RTS system at 25 • C, 6hr using the pIVEX2.4dvector and the hybrid ( vvsB inserted to pIVEX2.4dvector) plasmids .T he RTS products were subjected to SDS-PAGE.Lane M, Protein molecular weight marker (250,150,100,75,50,37,25,20,15, and 10 kDa); lane 1, pIVEX2.4dvector; lane 2, His-tag purification of pIVEX2.4dvector expressed protein; lane 3, circular vvsB (11.7 kDa, SGSHHHHHHSSGIEG); lane 4, His-tag purification of circular vvsB expr essed pr otein.thesized b y R TS.The N-terminal amino acid sequencing identified the sequence SGSHHHHHHSSGIEG, indicating the presence of the histidine-tagged VvsB.T hus , the expression of VvsB was confirmed.

The role of VvsB in in vivo system during the synthesis of serine protease VvsA and later on its proteolytic activity
We emplo y ed the tr aditional a ppr oac h of pr otein expr ession within the bacterial system to investigate the function of VvsB.In Fig. 1 B, a schematic of the genes inserted into the pBluescript IIKS + vector was presented.The constructs were generated with and without O/P and SD sequence derived from V. vulnificus NCIMB2137 (Fig. 1 B).
Subsequentl y, pr otein fr actions wer e extr acted fr om these r ecombinants, follo w ed b y the measur ement of pr oteol ytic activity.While the periplasmic pr otein fr action exhibited no activity (data not shown), the cytoplasmic fraction displayed pr oteol ytic activity (Fig. 3 ).Notabl y, the pr otein pr oduct fr om the vvsA gene exhibited higher activity compared to the protease derived from the vvsAB gene (Fig. 3 , lanes 3 and 4).
We found that the recombinants including O/P and SD deriv e fr om V. vulnificus NCIMB2137 (Fig. 3 , lane 3 and 4) sho w ed higher activity as compared to the constructs without these elements (Fig. 3 , lane 1 and 2).This result suggests that V. vulnificus NCIMB2137 specific O/P and SD sequence was essential for the pr oduction and activ ation of VvsA.It also indicates that the way VvsB might controls VvsA production is specific for V. vulnificus that could not be possible to mimic in other bacterial system such as E. coli (Kawase et al. 2022 ).
Next, we tried to measure the protease activity of protein produced from E. coli recombinants, in the presence of purified 5 ng or 100 ng VvsB (Fig. 4 ).E. coli recombinants containing O/P and SD sequences derived from V. vulnificus NCIMB2137, which had higher pr otease activity, wer e used.The r esults sho w ed that the recombinant VvsA had higher activity than the recombinant VvsAB ( t -test for comparison at eac h concentr ation shows significant difference at P < 0.05).The serine protease activity was observed to decrease gr aduall y with the addition of purified VvsB to the vvsA recombinant.Ho w e v er, this r eduction was not v ery m uc h significant.The results also sho w ed a slight decrease in pr oteol ytic activity with the addition of ≥ 10-100 ng of purified VvsB.In contrast, coexpression of VvsA and VvsB resulted in a significant reduction in serine protease activity.This observation suggests that VvsB acts as a weak inhibitor of serine protease activity.

Effect of VvsB on proteolytic activity of VvsA in in vitro
Next, we investigated the impact of purified VvsB on the proteolytic activity of VvsA-containing culture supernatant from V. vul-Figur e 3. T he pr oteol ytic activity of pr oteins pr oduced fr om E. coli r ecombinants.Azocasein (5 mg/ml) was used as a substr ate for the measur ement of protease activity per protein amounts .T he purified protein solution after the reaction then mixed with an equal volume of 0.5 M NaOH and the absorbance was measured at 440 nm.One protease unit (PU) was defined as the amount of enzyme hydrolyzing 1 μg of azocasein in 1 min.Lane 1, pBluescript IIKS + vector; lane 2, VvsA produced by vvsA into the pBluescript IIKS + vector; lane 3, Both VvsA and VvsB produced by vvsAB into the pBluescript IIKS + vector; lane 4, VvsA produced by vvsA including O/P and SD derived from V .vulnificus NCIMB2137 inserted into the pBluescript IIKS + vector; lane 5, Both VvsA and VvsB produced by vvsAB including O/P and SD derived from V .vulnificus NCIMB2137 inserted into the pBluescript IIKS + vector.The data is the mean + S.D. of three or more experiments .T he asterisk ( * ) indicates the significant difference ( P < 0.05).
Figur e 4. T he effect of VvsB on the pr oteol ytic activity of proteins produced from E. coli recombinants.VvsA produced by vvsA including O/P and SD deriv ed fr om V .vulnificus NCIMB2137 inserted into the pBluescript IIKS + v ector.Both VvsA and VvsB pr oduced by vvsAB including O/P and SD deriv ed from V .vulnificus NCIMB2137 inserted into the pBluescript IIKS + vector.Azocasein (5 mg/ml) was used as a substrate for the measurement of protease activity per protein amounts .T he purified protein solution after the reaction was mixed with an equal volume of 0.5 M NaOH and the absorbance was measured at 440 nm.One protease unit (PU) was defined as the amount of enzyme hydrolyzing 1 μg of azocasein in 1 min.Lane 1, VvsA; lane 2, VvsA added 5 ng of the purified VvsB; lane 3, VvsA added 10 ng of the purified VvsB; lane 4, VvsA added 100 ng of the purified VvsB, lane 5, VvsA and VvsB; lane 6, VvsA and VvsB added 5 ng of the purified VvsB; lane 7, VvsA and VvsB added 10 ng of the purified VvsB; lane 8, VvsA and VvsB added 100 ng of the purified VvsB.The data is the mean + S.D. of three or more experiments.nificus NCIMB2137.As illustrated in Fig. 5 , the pr oteol ytic activity of the supernatant was assessed in the presence of 1 ng to 1 μg of purified VvsB.Sur prisingl y, pr oteol ytic activity was maximal at 5 ng of VvsB, suggesting that the addition of VvsB did not act as an extracellular inhibitor (Fig. 5 ).Ther efor e, it is suggested that VvsB inhibits VvsA activity intr acellularl y to prev ent autol ysis , while it ma y facilitate VvsA activation outside the cell.
Figur e 5. T he effect of the pr oteol ytic activity in culture supernatant added VvsB.Azocasein (5 mg/ml) was used as a substrate for the measurement of protease activity.One protease unit (PU) was defined as the amount of enzyme hydrolyzing 1 μg of azocasein in 1 min.The culture supernatant was collected at early stationary phase at 30 • C. The Vibrio vulnificus culture supernatant and each VvsB (RTS product 0.67 μg/ μl) mixture (100 μl) was measured at 30 • C for 3 h.Lane 1, no add; lane 2, VvsB 1 ng; lane 3, VvsB 5 ng; lane 4, VvsB 10 ng; lane 5, VvsB 100 ng; lane 6, VvsB 500 ng; lane 7, VvsB 1 μg.The data is the mean + S.D. of three or more experiments.

Discussion
The vvsA gene with vvsB gene constitutes an operon, where Shine-Dalgarno sequence of vvsB gene overlaps with the stop codon (TTA) of vvsA gene.Earlier we had reported that even though VvsB can be synthesized b y R TS system, active VvsA cannot be synthesized (Kawase et al. 2022 ).
In general, a chaperone is a protein that aids in the proper folding of an immature protein to become functionally active.Once the correct folding is achieved, the chaperone protein dissociates fr om the matur e pr otein (Eder et al. 1993 , Cunningham andAgard 2003 ).
Since VvsA and VvsB have signal peptides, it is likely that these two proteins associate in the periplasm.Ho w e v er, the dir ect interaction between VvsA and VvsB in periplasm has not been anal yzed yet.In contr ast to gener al notion, we speculate that the interaction between VvsB and mature VvsA results into suppression in the activity of VvsA.In support of this speculation, it was found that VvsB can bind to the active form of VvsA and suppress its activity (Fig. 3 ).This suggests that VvsB acts as a weak inhibitor, but it is necessary to r eseac h mor eov er.
In Fig. 3 , the vvsA and vvsAB genes were recombined into the pr otein expr ession v ector pBluescript IIKS + , and the effect on serine protease activity was examined.We also conducted a compar ativ e experiment with and without O/P and SD sequences deriv ed fr om V. vulnificus NCIMB2137.These results sho w ed that the serine protease activity was increased in the vvsA recombinant compared to the vvsAB recombinant (Fig. 3 , lanes 3 and 4).Furthermore, the activity was increased in the strain containing genes containing O/P and SD sequences derived from V. vulnificus NCIMB2137 (Fig. 3 , lanes 3 and 4).This also rev ealed that VvsB r egulates VvsA pr oduction, and that the Vibrio -derived O/P and SD sequences are required to increase VvsA activity.
VvsA is a bacterial serine pr otease belie v ed to primaril y degr ade host-deriv ed pr oteins surr ounding the bacteria into an ingestible form for nutrient acquisition (Lee et al. 1999 ).For VvsA to exhibit its enzymatic activity, it must be expressed at the a ppr opriate site.Ho w e v er, the surviv al of the bacterium might get threatened if serine proteases accumulate excessively in inappropriate locations, such as the periplasm, leading to high protease activity.To avoid such circumstance, the le v el of pr otease expr ession and its activity must be tightly regulated.In case of VvsA, this intracellular c hec k on its expr ession le v el might be contr olled thr ough the inhibitory role of VvsB.Ho w e v er, this same VvsB seemed to act differ entl y in the extracellular milieu that leads to the production of active VvsA (Fig. 5 ).
Based on our findings, we propose that VvsA may undergo secretion into the extracellular environment in an intermediate state, retaining its C-terminal region.This intermediate form is then likely activated into its functional, active state with the assistance of VvsB.Ho w e v er, further r esearc h is necessary to thor oughl y understand the pr ecise mec hanisms gov erning this process.

Conclusions
Based on the r esults obtained, the r ole of VvsB in the activation of VvsA can be elucidated by the following hypothesis (Fig. 6 ): at first, VvsA tr ansits thr ough the outer membrane as an inactive intermediate of 59 kDa, then binds to the outer membrane surface and gr aduall y conv erts into the activ e form of 45 kDa VvsA, as evidenced by its activity in the cellular membrane fraction which was explained by the activity in the cellular membrane fraction.3 ).Ad ditionally, the acti ve form of VvsA undergoes rapid inactivation thr ough degr adation (Kaw ase et al. 2022 ), but it didn' t inhibit active VvsA and increased the serine protease activity (Fig. 5 ).Therefore, it was revealed that VvsB does not act as an inhibitor of VvsA activity in extracellular.Ho w ever, given that VvsB is presumably an outer membrane protein, this experimental system was solely designed for the functional analysis of VvsA.The route shown by the solid line in Fig. 6 is obtained from the current results, while the dotted line r epr esents the anticipated pathway.

Figure 6 .
Figure 6.Sc hematic r epr esentation for the pr ocess of formation of activ e VvsA and action of VvsB in the pr ocess